【作者】 李玲；

【导师】 潘兹书；

【作者基本信息】 武汉大学 ， 生物学、微生物学， 2015， 博士

【摘要】 猪瘟病毒(classical swine fever virus, CSFV)是猪的高致死性、烈性传染病——猪瘟(classical swine fever, CSF)的病原体。猪瘟的发生和流行对猪养殖业造成重大的经济损失。CSFV和牛病毒性腹泻病毒(bovine viral diarrhea virus, BVDV)、羊边界病病毒(border disease virus, BDV)同属于黄病毒科(Flaviviridae)瘟病毒属(Pestivirus)。CSFV的基因组为长度约12.3kb的单股正链RNA,包含两端的非编码区(untranslated region, UTR)和中间一个大的开放阅读框(open reading frame, ORF)。ORF编码一个大的多聚蛋白,该多聚蛋白经宿主细胞和病毒编码的蛋白酶水解加工产生具有功能的成熟蛋白,参与完成病毒的生命周期。作为RNA病毒研究的一个重要平台,反向遗传操作技术在CSFV基因组结构功能研究以及疫苗研发等方面具有重要作用。我们利用猪的RNA聚合酶Ⅰ(polⅠ)启动子驱动细胞内DNA转录产生RNA的特性,构建了一个新型的拯救CSFV反向遗传操作系统。将猪RNA pol Ⅰ启动子序列插入CSFV基因组cDNA的5’末端,鼠polⅠ终止子序列插入其3’末端,成功构建了CSFV石门株和猪瘟疫苗C株的全长cDNA感染性克隆pSPTI/SM和pSPTI/C。CSFV基因组cDNA在宿主细胞内利用细胞pol Ⅰ驱动猪pol Ⅰ启动子起始转录以及鼠的pol Ⅰ终止子终止转录,合成病毒基因组RNA,该vRNA没有5’端加帽修饰及3’poly(A)尾,具有精确的5’UTR和3’UTR末端；同时vRNA可作为mRNA直接翻译、加工得到病毒蛋白。分别将pSPTI/SM和pSPTI/C直接转染PK-15细胞,拯救出相应的CSFV强毒石门株和猪瘟疫苗C株病毒。这种基于polⅠ启动子的反向遗传操作系统能产生具有精确末端基因组的CSFV,且具有更高的拯救效率。以猪瘟疫苗C株的cDNA感染性克隆pSPTI/C为骨架,用强毒石门株UTR替换猪瘟疫苗C株的对应区域,构建获得了嵌合重组病毒株vC/SM 5UTR、 vC/SM3’UTR和vC/SMUTRs。特性研究表明,与猪瘟疫苗C株相比,石门株非编码区替换显著增强了重组嵌合病毒的复制能力,其复制效率由高至低依次为vC/SMUTRs、vC/SM3UTR和vC/SM 5’UTR;且重组病毒在PK-15细胞上形成蚀斑的能力也显示出与其病毒复制效率一致的趋势。嵌合重组病毒在PK-15细胞连续传代后其生长特性保持稳定。CSFV非结构蛋白NS2作为病毒编码的一种自切割半胱氨酸蛋白酶,对NS2-3前体蛋白之间的不完全加工而释放复制复合物关键因子NS3,NS2通过调节NS2-3的切割效率进而调节病毒基因组的复制。为了研究NS2蛋白在CSFV生命周期中的作用,利用反向遗传操作进行特异性位点突变分析,我们重点探讨了NS2 N端跨膜区的结构与功能。结果表明,NS2 N端NS2/D60A, NS2/D60K和NS2/D78K特异性位点突变完全妨碍了CSFV感染性病毒产生；NS2/R100A突变显著降低感染性病毒滴度；NS2/T37A、NS2/K52A、NS2/D78A及NS2/W85A突变不影响产生病毒的能力。通过构建单顺反子复制子研究证实,NS2/D60A、 NS2/D60K和NS2/D78K位点突变导致病毒基因组丧失复制能力,NS2/R100A使基因组复制效率显著降低,NS2/T37A、NS2/K52A、NS2/D78A和NS2/W85A基因组复制效率与野生型一致。表达Rluc活性重组病毒vA187-Rluc和vA187-Rluc/R100A特性分析与复制子结果显示,NS2通过调节病毒基因组RNA复制进而影响感染性病毒的产生。体外NS2-3前体蛋白生化分析结果显示,NS2 N端氨基酸点突变不影响NS2-3的切割效率和NS2-NS2自身的相互作用及蛋白稳定性；双顺反子复制子报告系统分析发现,NS2蛋白对病毒基因组的复制具有负调节作用；NS2氨基酸对基因组复制的调节作用不依赖NS2-3前体蛋白的加工效率。将致死突变体体外转录产物RNA转染细胞,经细胞连续传代获得感染性病毒。全基因组序列分析显示,病毒的恢复在于NS2/D60A和NS2/D60K位点的回复突变或NS2/D78K拟回复突变为NS2/K78E; NS2/R100A突变体的第二位点补偿突变NS2／I90L也导致病毒滴度达到野生型水平,暗示CSFV NS2 N端跨膜结构域之间存在相互作用。我们的研究成果为进一步理解CSFV基因组结构与功能、致病分子机制和发展基因工程疫苗奠定了坚实的基础。更多还原

【Abstract】 Classical swine fever is an economically important, highly contagious disease of pigs caused by the classical swine fever virus (CSFV). CSFV belongs to the genus Pestivirus within the family Flaviviridae, together with bovine viral diarrhea virus (BVDV) and border disease virus (BDV). The genome of CSFV is a single1 positive-strand RNA of about 12.3 kb which contains 5’-untranslated region (5’UTR), 3’UTR and a single large open reading frame (ORF). The ORF codes a polyprotein of approximately 3,898 amino acids, which is processed co-and post-translationally by host cell and viral proteases to produce structural and nonstructural proteins.Reverse genetics system is an important platform to study the structure and function of RNA viruses. Reverse genetics system allows to investigate molecular genetic basis of RNA virus replication, assembly and release of viral particle and pathogenesis by genome modification. In this study, a novel plasmid-based single step reverse genetics system for CSFV was developed. The recombinant plasmid harboring CSFV strain Shimen (pSPT1/SM), or Chinese (C) strain (pSPT1/C) full-length cDNA flanked by a swine RNA pol I promoter and murine pol I terminator was constructed, respectively. After transfection with the recombinant plasmid, an infectious CSFV with accurate ends was gererated in the transfected-PK15 cells. Compared with parental CSFV or vSM from in vitro transcripts RNA system, the CSFV rescued from the novel recombinant plasmid maintained same growth characteristics and plaque formation. The novel plasmid-based single step reverse genetics system for CSFV contributes to investigate molecular genetic basis of replication, assembly and release and pathogenesis of CSFV and the development of modified CSFV live vaccines, and provides potential basis for other pestiviruses.To understand the effect of UTR from highly virulent Shimen strain on viral replication of vaccine C-strain, several recombinant chimeric plasmids pC/SM UTRs, pC/SM 5’UTR and pC/SM 3’UTR were constructed based on the infectious cDNA clone pSPT1/C. The chimeric CSFVs were rescued from PK15 cells transfected with recombinant plasmids and characterization of the chimeric CSFVs was further evaluated. The results demonstrated that compared with CSFV C strain, the recombinant chimeric CSFVs containing the UTRs of the Shimen strain exhibited higher replication efficiency. Among them, vC/SM UTRs containing both 5’UTR and 3’UTR replicated higher than that of vC/SM 3’UTR, or vC/SM 5’UTR. After serial passages in PK-15 cells, the chimeric recombinant CSFVs maintained similar characteristics with parental recombinant viruses.As a CSFV-encoded autoprotease, NS2 creates in cis its own C terminus associating with a cellular chaperone termed Jiv or its fragment Jiv90 and thereby releases an essential viral replication complex component NS3. However, the additional function of cleaved NS2 for CSFV replication regulation is unknonwn. To understand the function for CSFV replication regulation, a series of mutations in NS2 N-terminus were constructed and the effect of the mutaions on infectious virus production and viral genome RNA replication was investigated. Our results showed that two aspartic acids mutations NS2/D60A, NS2/D60K and NS2/D78K abolished infectious virus production and the mutation NS2/R100A significantly decreased virus production. The mutantsNS2/T37A, NS2/K52A, NS2/D78A and NS2/W85A displayed similar virus titers compared with wt CSFV. The results based on the monocistronic replicon showed that no Rluc activity was detected in cells electroporated with the replicon containing NS2/D60A, NS2/D60K and NS2/D78K, and Rluc activities of NS2/R100A were significantly reduced at 72 h post-electroporation. In addition, the genomic RNA copies of the rescued vCSFV-Rluc and vCSFV-Rluc/R100A were detected using qRT-PCR after infection. The data showed that mutant NS2/R100A showed significantly reduced viral RNA copies, to the similar Rluc activities after infection viruses. These results suggested that the effect of NS2 N-terminal mutations on impairment of infectious virus production mainly resulted from the regulation of viral genome RNA replication level.Western blot analysis showed NS2-3 proteins containing wt and mutated NS2s had similar cleavage efficiency in vitro, and the interaction between NS2-NS2 and the stability of NS2 protein were not affected by the mutations. The correlation between cleavage efficiency of NS2-3 and viral RNA replication was further investigated based on the bicistronic replicon. Compared with NS3-NS5B/Rluc, reduced Rluc activity was observed from replicon NS2-NS5B/Rluc. Mutant replicon analysis showed that the RNA replication regulated by NS2 N-terminal mutations based on monocistronic replicon was similar to bicistronic replicons. These results demonstrated that the single amino acid within NS2 N-terminus modulated the viral RNA replication independently of the cleavage efficiency of precursor NS2-3.The serial passages of PK15 cells transfected with mutated CSFV genomic RNAs produced infectious virus particles. The genome sequencing exhibited that revertants of the NS2/D60A, NS2/D60K or NS2/D78K were obtained from passaged viruses. After a serial passages, the reduced infectious virus titer of mutant NS2/R100A was reversed by NS2/I90L compensatory second-site mutation, suggesting a possible interaction between transmembrane regions of NS2.Our works will contribute to explore the structure and function of CSFV genome, pathogenicity, and to develop CSFV genetically engineered vaccines.更多还原